An important accomplishment of the Program Project was to show that mutations in genes that participate in selected DNA repair pathways accelerate aging and/or cancer phenotypes in mice. In some cases, accelerated aging and/or increased cancer susceptibility were accompanied by an increase in somatic mutations. In other cases, accelerated aging occurred without an increase in mutations, suggesting other processes are responsible for the aging phenotypes. In all cases, however, we do not yet fully understand the cellular bases for the organismal phenotypes, which will be crucial for understanding how premature aging develops and how aging phenotypes are postponed by genome maintenance systems. Specifically, how do defects in genome maintenance impact the behavior and fate of cells, and, ultimately, how do the cellular responses cause the organismal phenotypes? This project aims to link the organismal phenotypes resulting from changes in genome maintenance systems with specific cellular phenotypes, using cells cultured from mice and humans. Our overall goals are to 1) understand the cellular consequences of intact or impaired genome maintenance; 2) determine how mouse and human cells differ in their responses to genotoxic stress; and 3) test specific hypotheses and strategies for manipulating cellular responses to genotoxic stress, in anticipation of creating additional mouse models within the Program Project. To achieve these goals, we propose to test cells, primarily embryo fibroblasts (MEFs), from wild type and mutant mice for proliferative and apoptotic responses under physiological oxygen and oxidative stress. Cellular endpoints will include replicative and stress-induced cellular senescence, cell death, growth in semisolid medium, and oxidative DNA damage and DNA double strand breaks. For mice carrying the lacZ mutation reporter, we will also determine mutation frequencies and , where feasible, compare the phenotypes of human fibroblasts with those of MEFs. We will also manipulate the expression level or function of selected mediators of the mammalian stress response and/or anti-oxidant defense systems, and determine whether these manipulations alter the behavior or fate of the cells. The overall goal of these experiments will be to identify potential interventions that can alter cellular endpoints that correlate with aging, and ultimately might postpone aging phenotypes in vivo. Finally, we will determine the feasibility of identifying senescence-responsive genes that can be used to detect and follow senescent cells in vivo, and eventually eliminate senescent cells in mice, with the expectation that this manipulation will delay or ameliorate selected aging phenotypes in the intact organism.